Investigation of the catalytic and regulatory properties of cardiac muscle protein kinase will help to illuminate the nature of the molecular interaction of cyclic AMP with isolated enzymes. Becuase cyclic AMP appears to affect the activity of many enzymes and because it may be the key intermediate in the mechanisms of action of numerous hormones, the potential importance of understanding in depth the chemistry underlying the catalytic action and regulatory behavior of cyclic AMP-activated enzymes like protein kinase may be very great. Our work on cyclic AMP-dependent protein kinase has the following specific aims for the next project period: (a) identification of curcial functional groups in the catalytic site; (b) further elucidation of the process by which the gamma-phosphoryl group of ATP is transferred to a peptide substrate; (c) determination of the conformation of peptide substrates bound to the catalytic subunit; and (d) determination of the environment at the sites of binding of cyclic AMP to the type II regulatory subunit and of the nature of the interactions between the regulatory and catalytic subunits. Additionally, we propose to extend our investigations of protein kinase action to the cyclic GMP-dependent enzyme. The approaches to be used are similar to those being successfully employed with cyclic AMP-dependent protein kinase and should lead to the formulation of a mechanism of action for the cyclic GMP-dependent enzyme.